Enzymatically amplified antibody assays have been successfully used to specifically monitor the expression of protein gene products in 96 well microtiter dish formats. See The Enzyme Linked Immunosorbent Assays (ELISA), Voller, A., Bidwell, D. E. and Bartlett, A. (1979) ISBN 0-906036.01.1. However, attempts to similarly monitor mRNA gene products in a 96 well microtiter format have failed due to the lack of sensitivity of the methods employed and the lack of facile mRNA isolation procedure. Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl, EDS., Greene Publishing Associates and Wiley-Interscience (1987). Further, the up and/or down regulation of a particular gene can be indirectly measured by reporter gene constructs which rely on the heterologous expression of a gene product that is capable of subsequent detection. Current Protocols in Molecular Biology, Supra. While the latter approach is useful, it suffers from several limitations. Those limitations include the need to prepare, identify and characterize appropriate constructs; the constructs comprise a heterologous promoter and a reporter gene which necessitates that the promoter be available and characterized; promoter activity only is measured; use of enzymes to measure activity means that translation and/or enzyme inhibitors can compromise the integrity of the assay; and integration of the reporter gene may not be targeted to the natural chromosomal site and often times multiple copies per cell result which can influence gene regulation.
A method which utilizes gene specific oligomers to provide specificity for a particular gene and particular DNA polymerases to amplify the specific gene sequences to detectable levels, a method generally known as polymerase chain reaction or PCR, is described by Saiki, R., et al., Science 230: 1350 (1985) and Saiki, R. K., et al., Science 239: 487 (1988).
It has been reported that mRNA could be detected from cells cultivated in vitro in a well of a 96 well microtiter dish. Russell Higuchi, Simple and Rapid Preparation of Samples for PCR in PCR Technology, Henry A. Elrich, Ed., M. Stockton Press (1989). However, the mRNA isolation method employed therein is not conducive to the utilization of PCR in screening processes, particularly when such screening involves a plurality of samples.